CN113880927A - 通过过表达锌指蛋白OsCIP3增强水稻低温耐受性的方法 - Google Patents
通过过表达锌指蛋白OsCIP3增强水稻低温耐受性的方法 Download PDFInfo
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Abstract
本发明公开了通过过表达锌指蛋白OsCIP3增强水稻低温耐受性的方法。本发明提供了OsCIP3蛋白或其相关生物材料在调控植物低温耐受性中的应用;所述相关生物材料为能够表达所述OsCIP3蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。本发明实验显示向中花11中导入OsCIP3蛋白的编码基因后植株的低温耐受性提高;而降低中花11中OsCIP3的表达后植株的低温耐受性明显降低。证明OsCIP3蛋白及其编码基因可以调控植物的低温耐受性性,对于培育植物耐低温新品种具有重要意义。
Description
技术领域
本发明涉及基因工程领域,具体涉及通过过表达锌指蛋白OsCIP3增强水稻低温耐受性的方法。
背景技术
水稻(Oryza sativa L.)起源于热带亚热带地区,最适温度为25–30℃,相对于小麦、燕麦等作物更易受到低温胁迫(Zhang Q,Chen QH,Wang SL,et al.Rice and coldstress:methods for its evaluation and summary of cold tolerance-relatedquantitative trait loci.Rice:2014,7:24-26.)。根据不同温度范围内起作用的温度和各种生理机制,植物中的低温胁迫可以分冷害(chilling damage)和冻害(freezinginjury)两大类。冷害是指0–20℃的低温伤害,在热带和亚热带地区,冷害是最主要的低温胁迫,植物对这种伤害的适应性叫抗冷性(Suh JP,Jeung JU,Lee JI,etal.Identification and analysis of QTLs controlling cold tolerance at thereproductive stage and validation of effective QTLs in cold-tolerantgenotypes of rice(Oryza sativa L.).Theoretical and Applied Genetics:2010,120:985–995.;Xu LM,Zhou L,Zeng YW,et al.Identification and mapping ofquantitative trait loci for cold tolerance at the booting stage in a japonicarice near-isogenic line.Plant Science:2008,174:340–347.);冻害是指0℃以下的低温环境使作物体内结冰,从而对作物造成的伤害。在温带气候区域,低温胁迫可以诱导冷驯化,使得植物适应这种伤害从而抵御低温,植物对这种伤害的适应性叫做抗冻性。低温胁迫在水稻生长发育的不同时期危害性大不相同(Jena,KK,Kim,et al.Identification ofcold-tolerant breeding lines by quantitative trait loci associated with coldtolerance in rice.Crop Science:2012,52:517–523.)。水稻在发芽期遭遇低温胁迫会严重降低出芽率,亦导致烂秧死苗;而苗期温度低于15℃便会影响水稻的生长(Zhang Q,ChenQH,Wang SL,et al.Rice and cold stress:methods for its evaluation and summaryof cold tolerance-related quantitative trait loci.Rice:2014,7:24-26.),长期受低温胁迫后,叶片失绿泛黄,卷曲呈枯萎状,严重时会产生黄色条状斑纹;孕穗期低于17℃会减少颖花数、抑制小穗发育、延长抽穗天数并且降低穗抽出度(Zhang ZY,Li JH,Li F,etal.OsMAPK3 phosphorylates OsbHLH002/OsICE1 and inhibits its ubiquitination toactivate OsTPP1 and enhances rice chilling tolerance.Developmental Cell:2017,43(6):731–743);开花期持续遭遇低温,会降低受精结实率造成空壳瘪粒,直接影响最终产量。当胁迫来临时,植物虽然不能像动物一样通过移动来躲避,但却可以通过改变内部调控网络使自己免受迫害。长期以来,水稻就在自然或人工选择下进化出了应对低温胁迫的复杂机制(Chen L,Zhao Y,Xu S,et al.OsMADS57 together with OsTB1 coordinatestranscription of its target OsWRKY94and D14 to switch its organogenesis todefense for cold adaptation in rice.New Phytologist:2018,218:219–231.)。然而对于水稻低温信号调控机制的分子机制的挖掘也只是冰山一角,因此了解水稻对低温胁迫的抵御机制对改善水稻耐低温的能力,提高作物产量具有重要意义。
水稻中低温信号转导主要有三种途径:第一种是ABA参与调控的信号网络。冷胁迫下,水稻体内ABA积累,增加ABA响应元件(ABRE)与ABF的结合效率(Ding YL,Shi YT,YangSH.Advances and challenges in uncovering cold tolerance regulatory mechanismsin plants.New Phytologist:2019,222:1690–1704.)。ABF是一类bZIP型转录因子可以调控下游NAC基因表达进而参与冷胁迫调控。在拟南芥中主要是以PYR/PYL/RCAR-PP2C-SnRK2为核心构成ABA冷胁迫响应途径(Wang HJ,Tang J,Liu J,et al.Abscisic acidsignaling inhibits Brassinosteroid signaling through dampening thedephosphorylation of BIN2 by ABI1 and ABI2.Molecular Plant:2018,11:315–325.;Zhang Q,Kong XG,Yu Q,et al.Responses of PYR/PYL/RCAR ABA receptors tocontrasting stresses,heat and cold in Arabidopsis.Plant Signaling&Behavior:2019,14(12):1670596–1670601.)。低温胁迫下,ABA-PYR-PP2C以复合体形式存在,阻断SnRK2与PP2C的结合,游离SnRK2通过自磷酸化激活,并且磷酸化下游bZIP转录因子(LiuCT,Ou SJ,Mao BG,et al.Early selection of bZIP73 facilitated adaptation ofjaponica rice to cold climates.Nature communications:2018,9:3302–3314.)及其他转录因子,进行冷胁迫调控(Guo XY,Liu DF,Chong K Cold signaling in plants:Insights into mechanisms and regulation.Journal of Integrative Plant Biology:2018,60:745–756.;Zhu J K.Abiotic stress signaling and responses inplants.Cell:2016,167:313–324.);第二种是钙离子途径。冷害来临时,激活膜上钙离子通道导致胞内钙离子浓度增加,OsCaM、OsCBL等蛋白以及钙离子依赖的蛋白激酶CDPKs快速感知钙离子并促进OsMYB3R-2、OsDREB2A等基因的表达,其产物通过结合COR基因的启动子上的CRT/DRE调控元件来调控COR基因的表达,从而提高水稻耐冷性(Zhu J K.Abioticstress signaling and responses in plants.Cell:2016,167:313–324.);第三种是ROX途径。水稻受到低温胁迫,细胞内氧代谢平衡失调产生大量的ROS。ROS促进OsMKK6的表达(Xie G,Kato H,Imai R.Biochemical identification of the OsMKK6-OsMPK3signalingpathway for chilling stress tolerance in rice.Biochemical Journal:2012,443:95–102.),OsMKK6促进OsMAPK3表达并使其磷酸化,磷酸化的OsMAPK3进入细胞核一方面抑制OsbHLH002/OsICE1被HOS1降解,另一方面与OsbHLH002/OsICE1互作,激活OsTPP1的表达进而增强水稻耐冷性(Guo XY,Liu DF,Chong K Cold signaling in plants:Insightsinto mechanisms and regulation.Journal of Integrative Plant Biology:2018,60:745–756.;Zhang ZY,Li JH,Li F,et al.OsMAPK3phosphorylates OsbHLH002/OsICE1 andinhibits its ubiquitination to activate OsTPP1 and enhances rice chillingtolerance.Developmental Cell:2017,43(6):731–743)。此外Lee等发现PhyB在拟南芥中借助PIF4、PIF7互作因子可与CBFs结合,进而调控低温信号(Lee CM,MichaelF.Thomashow.Photoperiodic regulation of the C-repeat binding factor(CBF)coldacclimation pathway and freezing tolerance in Arabidopsisthaliana.Proceedings of the National Acad Sciences:2012,109:15054–15059.)。He等发现水稻中phyB突变体低温耐受性增强,表明PhyB负调控水稻耐冷性(He Y,Li Y,CuiL,et al.Phytochrome B negatively affects cold tolerance by regulating OsDREB1gene expression through phytochrome interacting factor-like protein OsPIL16in rice.Frontiers in Plant Science:2016,7:1963–1975.)。
锌指蛋白(ZFP)最早在非洲爪蟾的卵母细胞中发现,是一类具有指状结构域的转录因子,锌指蛋白在植物中非常丰富且具有多种功能,包括DNA结合功能和转录调节功能,与真核生物的生长发育和抗逆相关。锌指蛋白因其结构与特征得名。在锌指蛋白中,几个保守的氨基酸(一般为半胱氨酸Cys与组氨酸His)与一个Zn2+结合,形成一个相对独立的区域。根据结构域中半胱氨酸和组氨酸的残基数目和围绕Zn2+所形成的空间结构,锌指蛋白可以分为:C2H2、C2C2和C3H等亚类。其中,C2H2型(也称TFIIIA型)ZFP是研究最多也是最为深入的一类(Xiang J,Li L,Chen X.Progress in the study of abiotic stress-relatedzinc finger protein genes in plant.ActaAgriculturaeNucleataeSinica:2012,26:666–672,716.;CiftciYilmaz S,Mittler R.The zinc finger network ofplants.Cellular and Molecular Life Sciences:2008,65:1150–1160.),目前为止,已经报道了包括矮牵牛,拟南芥,小麦和水稻在内的植物中的C2H2锌指蛋白,且这些蛋白中的大多数在锌指结构域中具有植物特异的QALGGH基序(Huang J,Wang JF,Zhang HS.Structureand function of plant C2H2 zinc finger protein.Yi chuan=Hereditas:2004,26:414–418.)。C2H2型锌指由两个Cys和两个His组成,与Zn2+、α-螺旋和反向平行的双链β-折叠一起形成保守核心基序,因此C2H2锌指结构是一个稳定且相对独立的蛋白质结构域,C2H2型锌指蛋白在植物对各种胁迫的应答中发挥着重要的作用,比如低温,盐,干旱,氧化应激等(Yue X,Que Y,Xu L,et al.ZNF1 Encodes a putative C2H2 zinc-finger proteinessential for appressorium differentiation by the rice blast fungusmagnaportheoryzae.Molecular Plant-Microbe Interactions:2016,29:22–35.;Muthamilarasan M,Bonthala VS,Mishra AK,et al.C2H2type of zinc fingertranscription factors in foxtail millet define response to abioticstresses.Functional&Integrative Genomics:2014,14:531–543.;Kim JC,Lee SH,Cheong YH,et al.A novel cold-inducible zinc finger protein from soybean,SCOF-1,enhances cold tolerance in transgenic plants[J].Plant Journal,2001,25:247–259.;Wang LJ,He SZ,Zhai H,et al.Molecular cloning and functionalcharacterization of a salt tolerance-associated gene IbNFU1 fromSweetpotato.Journal of Integrative Agriculture:2013,12:27–35.)。
发明内容
本发明的目的是提供一种通过过表达锌指蛋白OsCIP3增强水稻低温耐受性的方法。
第一方面,本发明要求保护OsCIP3蛋白或其相关生物材料在调控植物低温耐受性中的应用。
所述相关生物材料为能够表达所述OsCIP3蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
所述OsCIP3蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
上述蛋白质中,所述标签是指利用DNA体外重组技术,与目的蛋白一起融合表达的一种多肽或者蛋白,以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中,同源性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
上述蛋白质中,所述95%以上的同源性可为至少96%、97%、98%的同一性。所述90%以上的同源性可为至少91%、92%、93%、94%的同一性。所述85%以上的同源性可为至少86%、87%、88%、89%的同一性。所述80%以上的同源性可为至少81%、82%、83%、84%的同一性。
在所述应用中,所述OsCIP3蛋白在所述植物中的表达量和/或活性提高,所述植物的低温耐受性提高。所述OsCIP3蛋白在所述植物中的表达量和/或活性降低,所述植物的低温耐受性降低。
第二方面,本发明要求保护一种培育低温耐受性改变的植物品种的方法。
本发明所要求保护的培育低温耐受性改变的植物品种的方法,可为如下方法A1或方法A2:
方法A1:一种培育低温耐受性提高的植物品种的方法(或称为“提高植物低温耐受性的方法”),可包括使受体植物中OsCIP3蛋白的表达量和/或活性提高的步骤。
方法A2:一种培育低温耐受性降低的植物品种的方法(或称为“降低植物低温耐受性的方法”),可包括使受体植物中OsCIP3蛋白的表达量和/或活性降低的步骤。
其中,所述OsCIP3蛋白为前文(A1)-(A4)中任一所示蛋白。
第三方面,本发明要求保护一种培育低温耐受性改变的转基因植物的方法。
本发明所要求保护的培育低温耐受性改变的转基因植物的方法,可为如下方法B1或方法B2:
方法B1:一种培育低温耐受性提高的转基因植物的方法,包括如下步骤:向受体植物中导入能够表达OsCIP3蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比低温耐受性提高。
方法B2:一种培育低温耐受性降低的转基因植物的方法,包括如下步骤:对受体植物中能够表达OsCIP3蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比低温耐受性降低。
其中,所述OsCIP3蛋白为前文(A1)-(A4)中任一所示蛋白。
在所述方法B1中,向所述受体植物中导入能够表达所述OsCIP3蛋白的核酸分子可通过任何能够实现这一目的的技术手段实现。如能够表达所述OsCIP3蛋白的核酸分子可通过重组载体的形式导入所述受体植物中。
在本发明中,所述重组载体中启动能够表达所述OsCIP3蛋白的核酸分子转录的启动子为玉米泛素启动子。所述玉米泛素启动子的核苷酸序列可如EQ ID No.3第1~1987位所示。
在上述方法B2中,对所述受体植物中能够表达所述OsCIP3蛋白的核酸分子进行抑制表达可通过任何能够实现这一目的的技术手段实现。如利用CRISPR/Cas9系统基因编辑目的植物从而抑制OsCIP3基因的表达。在本发明的具体实施方式中,CRISPR/Cas9的靶序列具体为SEQ ID No.4和SEQ ID No.5。
在所述方法中,将携带有所述核酸分子的所述重组载体或者用于对所述受体植物中所述核酸分子进行敲除或抑制表达时采用的基因编辑工具导入所述受体植物,具体可为:通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织,并将转化的植物组织培育成植株。
在上述各方面中,能够表达所述OsCIP3蛋白的核酸分子可为如下任一所述的DNA分子:
(a1)SEQ ID No.2所示的DNA分子;
(a2)在严格条件下与(a1)限定的DNA分子杂交且编码所述OsCIP3蛋白的DNA分子;
(a3)与(a1)-(a2)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述OsCIP3蛋白的DNA分子。
上述核酸分子中,所述严格条件可为如下:50℃,在7%十二烷基硫酸钠(SDS)、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,2×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.5×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在65℃,0.1×SSC,0.1%SDS中漂洗;也可为:在6×SSC,0.5%SDS的溶液中,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
上述核酸分子中,同源性是指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算,然后即可获得同一性的值(%)。
上述核酸分子中,所述95%以上的同源性可为至少96%、97%、98%的同一性。所述90%以上的同源性可为至少91%、92%、93%、94%的同一性。所述85%以上的同源性可为至少86%、87%、88%、89%的同一性。所述80%以上的同源性可为至少81%、82%、83%、84%的同一性。
在上述各方面中,所述植物可为单子叶植物。
进一步地,所述单子叶植物可为禾本科植物。
更进一步地,所述禾本科植物可为水稻。
在本发明的具体实施方式中,所述水稻具体为水稻品种中花11。
实验证明,向中花11中导入OsCIP3蛋白的编码基因后植株的低温耐受性提高;利用CRISPR/Cas9系统降低中花11中的OsCIP3基因的表达可以减弱水稻低温耐受性。证明OsCIP3蛋白及其编码基因可以调控植物的低温耐受性,对于培育植物耐低温新品种具有重要意义。
附图说明
图1为OsCIP3超表达株系中OsCIP3表达量检测。A为pUN1301-OsCIP3载体构建示意图;B为Q-PCR检测OsCIP3超表达株系中OsCIP3的表达量。
图2为OsCIP3基因编辑突变体的分子鉴定。A为pCRISPR-OsCIP3载体构建示意图;B为ZH11与oscip3-2和oscip3-3编辑位点比对。
图3为OsCIP3转基因植株的低温耐受表型及统计图。A为生长到三叶期的ZH11、OsCIP3-OE8和OsCIP3-OE14幼苗冷处理前拍照;B为ZH11、OsCIP3-OE8和OsCIP3-OE14幼苗在4℃冷处理84小时,恢复生长一个月后拍照。C为B中存活率统计。Student’s t test用于差异显著性分析,所有数据为三次生物学重复的统计结果(n>30),**代表p<0.01。
图4为OsCIP3突变体低温处理前、后表型观察及统计图。A为生长到三叶期的ZH11、oscip3-2及oscip3-3幼苗冷处理前拍照;B为ZH11、oscip3-2及oscip3-3幼苗在4℃冷处理72小时,恢复生长一个月后拍照。C为B中存活率统计。Student’s t test用于差异显著性分析,所有数据为三次生物学重复的统计结果(n>30),**代表p<0.01。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、通过调节OsCIP3基因表达从而调控水稻低温耐受性
一、OsCIP3蛋白的编码基因OsCIP3的获得
根据水稻基因组数据库提供的基因信息,OsCIP3的基因号为Os01g65080,CDS长度为1569个核苷酸(SEQ ID No.2),含有2个外显子,1个内含子,编码522个氨基酸(SEQ IDNo.1),包含C2H2锌指蛋白结构域,具有转录激活活性。
根据数据库分析的结果设计引物,正向引物(BamH I):5′-CGGGATCCATGGACAGTGGCTTGGGAAGAA-3′,反向引物(Kpn I):5′-GGGGTACCGCTGTCTCCGTTCTGCTGCCCG-3′,提取粳稻中花11(Oryza sativa L.cv Zhonghua11,ZH11,公众可以从中国科学院植物研究所获得)的三叶期幼苗RNA,反转录为cDNA,采用PCR方法扩增到1569个核苷酸的全长编码区序列片段。
具体操作过程如下:
(1)植物RNA的提取:使用Magen公司的HiPure Plant RNA Mini Kit进行植物总RNA提取。使用的试剂、研钵、试管和枪头等均为RNase-free,防止RNase污染从而降解RNA。使用前在Buffer RW2中加入适量无水乙醇。选取0.5g三叶期中花11水稻的幼苗为材料,在液氮中研磨,加入500μL Buffer RL,涡旋震荡充分打散样品,室温静置3分钟;室温14000×g离心5分钟;将离心后的上清转移到gDNA Filter Column过滤柱中,过滤柱放入2mL收集管中,14000×g离心2分钟;在过滤后的溶液中加入250μL的无水乙醇,吹吸混匀;将700μL滤液转移至HiPure RNA Mini Column过滤柱中,过滤柱放入2mL收集管,14000×g离心1分钟,弃废液。多次转移中的溶液直至完全过滤;加入500μL Buffer RW1,12000×g离心1分钟,弃废液;加入500μL Buffer RW2,12000×g离心1分钟,弃废液;12000×g离心2分钟,去除残留的乙醇;将过滤柱转移至一个干净的1.5mL RNase-free离心管中,滴加30-50μL的RNase-freeddH2O至膜中央,室温静置2分钟后12000×g离心2分钟;将提取成功的RNA保存于-80℃备用。
(2)反转录:使用赛默飞公司的High Capacity cDNA Reverse TranscriptionKits进行反转录第一链cDNA的合成。所有操作均在冰上进行。取备用待反转录的RNA2μg,加RNase-free H2O至10μL;使用试剂盒内的试剂配置2×RT master mix,于冰上温和混匀,成分如表1。
表1反转录组分表
将2×RT master mix和RNA混合,按照25℃/10分钟,37℃/120分钟,85℃/5分钟,4℃/∞的条件进行反转录;将反转录得到的cDNA保存于-20℃备用。
(3)PCR扩增:提取的cDNA稀释10倍,用作模板按以下体系进行PCR反应:0.2μlPrimerSTAR HS DNA Polymerase(5U/μL)、10μL 2×GC buffer,1.8μL dNTPs,0.5μL 5′端引物(10μM),0.5μL 3′端引物(10μM),加ddH2O终体积20μL。引物序列5′端引物:5′-ATGGACAGTGGCTTGGGAAGAA-3′,3′端引物:5′-GCTGTCTCCGTTCTGCTGCCCG-3′,PCR程序为:98℃预变性30秒后进入PCR循环,循环参数为98℃/10秒变性→52℃/15秒复性→72℃/4分钟20秒延伸,35个循环后再72℃继续合成10分钟。
扩增的PCR产物经过0.8%的琼脂糖凝胶电泳分离后测序得到OsCIP3的CDS序列产物(SEQ ID No.2的第1-1566位)。
二、超表达载体pUN1301-OsCIP3的构建
1、pUN1301载体的获得
(1)剪取约0.2g玉米(品种名:中作-中单8,北京中农作科技发展有限公司)幼苗,置于液氮中研磨;然后加入800μL新配制的提取缓冲液(含0.1M Tris-HCl pH8.0,50mMEDTA,0.5M NaCl,1%SDS和1%β-巯基乙醇),剧烈振荡使其全部悬浮;65℃水浴30分钟,每5分钟颠倒混匀一次;然后加入250μL预冷的5M乙酸钾水溶液,立即颠倒混匀,冰浴5分钟;加入等量酚/氯仿,抽提一次,12000rpm离心5分钟;收集上清液,加入0.6倍体积的异丙醇沉淀DNA,室温放置40分钟;4℃12000rpm离心15分钟,弃上清;沉淀用70%、100%乙醇各洗一次;干燥后,溶于20μL含100μg/mL RNase的ddH2O中,得到玉米基因组DNA。
(2)取上述玉米基因组DNA溶液2μL作为模板,在带有Hind III识别位点的5′引物(5′-GGAAGCTTCTGCAGTGCAGCGTGACCCGG-3′)和带有BamHI识别位点的3′引物(5′-CGGGATCCAAGTAACACCAAACAACAGGG-3′)为引物,进行PCR扩增,PCR反应条件为:先94℃,3分钟;再94℃,45秒,62℃,45秒,72℃,2分钟,共35个循环,最后72℃,10分钟。反应结束后,对PCR产物进行0.8%琼脂糖凝胶电泳检测,表明得到长度约为2kb的扩增片段,与预期结果相符,回收该目的片段,得到的片段经测序验证,为玉米泛素启动子(UbiPro)。
上述玉米泛素启动子(UbiPro)也可以人工合成获得。
(3)用限制性内切酶Sac I和EcoR I将Noster poly A终止序列(277bp)从质粒载体pBI121(北京拜尔迪生物技术有限公司目录号:MP-091)上切下,连接到载体pUC19(北京百泰克生物技术有限公司目录号:DP7801)的Sac I和EcoR I位点间,得到重组载体,命名为pUC19-Noster。再用限制性内切酶HindIII和BamHI双酶切pUC19-Noster,琼脂糖凝胶电泳检测后,回收线性化的载体大片段,并将该回收片段与(2)中经Hind III和BamH I双酶切获得的带有粘性末端的玉米泛素启动子(UbiPro)相连,得到重组载体,命名为pUN19。
(4)用限制性内切酶EcoR I部分酶切和HindIII完全酶切(37℃条件下,先加入EcoR I进行部分酶切,酶切时间为半小时,65℃下20分钟使EcoR I酶失活,后加入HindIII完全酶切3小时即可)。从(3)构建的重组载体pUN19切下包含UbiPro和Noster的长度约为2.3kb的片段(该片段的核苷酸序列为SEQ ID No.3,其中,SEQ ID No.3第1~1987位为UbiPro序列,SEQ ID No.3第2026~2291位为Noster序列),将该片段克隆入质粒载体pCAMBIA1301(BiovectorCo.,LTD公司目录号Biovec-11)的EcoRI和HindIII位点处,得到重组载体,命名为pUN1301。
2、pUN1301-OsCIP3的构建
用限制性内切酶BamH I和Kpn I对1步骤获得的质粒pUN1301进行双酶切,酶切体系为:质粒2μL、10×酶切缓冲液2μL、BamH I 0.5μL(10U/μL)、KpnI 0.5μL(10U/μL),加ddH2O补充反应体系至20μL,37℃酶切1小时。用琼脂糖凝胶电泳对酶切产物进行分离,回收4392bp线性化的pUN1301大片段,溶于20μL ddH2O中。
将步骤一中获得的3μL OsCIP3基因CDS溶液、1μL回收的pUN1301大片段溶液以及5μl重组酶2×SoSoo Mix Plus(Tsingke Biological Technology,货号TSV-S2)混合,50℃孵育15min,得到的连接产物转化大肠杆菌DH5α感受态细胞,经含卡那霉素的抗性平板筛选得到阳性克隆。提取阳性克隆中的重组质粒,且测序验证正确,命名为pUN1301-OsCIP3。在该表达载体中采用玉米泛素启动子(UbiPro)启动目的片段OsCIP3在植物中超表达。pUN1301-OsCIP3载体构建示意图如图1中A所示。
三、OsCIP3基因pCRISPR-Cas9载体构建
1、SgRNA靶点设计与oligo序列合成
将OsCIP3基因的CDS序列(SEQ ID No.1)全长输入http://www.e-crisp.org/E-CRISP/designcrispr.html网站,参数按照以下设置:物种选择“Orzya sativa IRGSP-1.0.31”,输入格式选择“Input is FASTA sequence”,“Start application”选择“medium”,选择“Start SgRNA search”进行SgRNA序列搜索。选择保守SgRNA序列作为OsCIP3基因的SgRNA序列,设计合成2条sgRNA。
sgRNA1的靶序列:5′-TCAGGCAATGCTGAGCAAC-3′(SEQ ID No.4);
sgRNA2的靶序列:5′-CCCTGGGTCTTATGTAGTATTG-3′(SEQ ID No.5)。
2、重组载体pCRISPR-OsCIP3的构建
在靶序列的5’端加上TGTG作为正向引物,在靶序列的反向互补5’端加上AAAC作为反向引物,最后得到如下引物:
sgRNA1的合成引物:
Forward:5’-TGTGTCAGGCAATGCTGAGCAAC-3’;
Reverse:5’-AAACGTTGCTCAGCATTGCCTGA-3’。
sgRNA2的合成引物:
Forward:5’-TGTGCCCTGGGTCTTATGTAGTATTG-3’;
Reverse:5’-AAACCAATACTACATAAGACCCAGGG-3’。
将上述sgRNA1的2条合成引物(10μM)等体积混匀在PCR仪上进行Oligo二聚体制备,条件如下:95℃3分钟,然后以0.2℃/S的速度降到20℃,得到sgRNA1编码核酸(Oligo二聚体)。
将上述sgRNA2的2条合成引物(10μM)等体积混匀在PCR仪上进行Oligo二聚体制备,条件如下:95℃3分钟,然后以0.2℃/S的速度降到20℃,得到sgRNA2编码核酸(Oligo二聚体)。
取2μL BGK03载体(记载在如下文献中:Yuming Lu et al.,Genome-wideTargeted Mutagenesis in Rice Using the CRISPR/Cas9 System.Molecular Plant,2017Sep12;10(9):1242-1245.,公众可从申请人处获得,仅可用于重复本发明试验使用,不得他用),用Bas I酶进行酶切,酶切体系如下:
37℃酶切2小时,酶切产物用0.8%琼脂糖凝胶电泳进行分离,切下15,000bp线性化的BGK03大片段进行回收,最终产物30μL的ddH2O溶解,得到线性化的BGK03载体。
然后,将1μL线性化的BGK03载体分别与3μL sgRNA1编码核酸和sgRNA2编码核酸用T4连接酶连接,连接体系如下:
得到的连接产物转化大肠杆菌DH5α感受态细胞,经卡那霉素的抗性平板筛选得到阳性克隆。提取阳性克隆中的重组质粒,进行测序验证,得到含有OsCIP3基因的不同sgRNA编码序列和Cas9编码序列的CRISPR载体(图2中A),经测序验证正确后命名为pCRISPR-OsCIP3。
四、转基因水稻的获得
将上述pUN1301-OsCIP3、pCRISPR-OsCIP3质粒分别转化农杆菌EHA105(Hiei Y,Ohta S,Komari T,Kumashiro T(1994)Efficient transformation of rice(Oryzasativa L.)mediated by Agrobacterium and sequence analysis of the boundariesof the T-DNA.Plant J 6:271–282,公众可从中国科学院植物研究所获得),经含卡那霉素的抗性平板筛选得到阳性克隆的超表达工程菌,PCR鉴定阳性克隆。
将pUN1301-OsCIP3与pCRISPR-OsCIP3质粒分别侵染ZH11水稻的愈伤组织,将表达pUN1301-OsCIP3与pCRISPR-OsCIP3的农杆菌28℃,200rpm过夜震荡培养,离心收集菌液,弃上清。随后用AAM-AS培养基重悬菌体,愈伤侵染20分钟,将愈伤组织转到继代培养基N6D2C,暗培养3天,再将愈伤组织用含300mg/L头孢霉素的无菌水洗涤5遍,无菌滤纸吸干后转至N6D2S1培养基上,筛选一代;两周后,转移至N6D2S2培养基上筛选二代(2周/代);取出经过3代筛选生长旺盛的抗性愈伤组织,转移至分化培养基(1),上,在分化培养箱(12小时光周期,白天28℃,夜晚25℃)中培养7天;然后转移至分化培养基(2),上,在分化培养箱中培养至产生再生苗。再生的植株在生根壮苗培养基上生根壮苗;待小苗长至10厘米左右时,打开容器封口膜,炼苗2-3天,然后将小苗移入人工气候室栽培,获得T0代转基因水稻。
所用培养基如下表2。
表2所用培养基配方
实验同时设置向根癌农杆菌EHA105中导入pUN1301载体或者BGK03载体后侵染水稻中花11的空载对照。
五、转基因水稻的鉴定
1、超表达OsCIP3的水稻的鉴定
提取T2代转pUN1301-OsCIP3水稻幼苗的总RNA,经过RNase free DNase I处理2μg总RNA用M-MLV反转录酶进行反转录成cDNA第一条链。将植物总RNA反转录成cDNA,利用Primer Express 2.0程序(Applied Biosystems)设计基因特异引物,并以Actin引物为内标参照,Actin正向引物:5′-TGGTCGTACCACAGGTATTGTGTT-3′反向引物:5′-AAGGTCGAGACGAAGGATAGCAT-3′。Tm值为55–60℃,GC含量在40–60%之间,扩出的目的片段长度为100–150bp。用于定量PCR检测的引物如下:
正向引物:5′-GAGCCTCAAATTCTACTCGC-3′;
反向引物:5′-CTTCCCAAGCCACTGTCCAT-3′。
在实时定量PCR仪MX3000P(Stratagene,USA)上运行PCR程序,95℃2min;95℃15s,58℃10s,72℃15s;共45循环;95℃30s,58℃30s,95℃30s。根据CT值计算基因的相对表达量。
结果如图1中B所示,在Actin作为内参的情况下,与野生型水稻(ZH11)相比,Line5、7、8、9、11、13和14植株的T2代转OsCIP3水稻幼苗中OsCIP3基因的表达丰度有了不同程度的上调,说明目的基因OsCIP3在转录水平已经成功表达。其中OE8和OE14表达量相对较高,选取这两个株系进行表型实验。
2、OsCIP3基因CRISPR/Cas9突变体鉴定
提取DNA和PCR的方法如前所述,用于PCR检测的引物如下:
Target1的鉴定引物:
Forward:5’-CCTGGTCAATCGTTCCCTG-3’;
Reverse:5’-TCATCATCTCATCGCTTTCTGCC-3’。
Target2的鉴定引物:
Forward:5’-GCGATGAGATGATGAACTGC-3’;
Reverse:5’-GGCGAGAAATAGCGTGGA-3’。
对获得的目的片段进行测序分析,如图2中B所示,oscip3-2株系在第一个靶序列(Target1)处产生了4bp的碱基缺失,在第二个靶序列(Target2)处产生了3bp的碱基缺失。oscip3-3株系在Target1处插入了1bp的碱基,在Target2处产生了2bp的碱基缺失,从而造成了OsCIP3基因的突变。因此oscip3-2以及oscip3-3为OsCIP3基因的功能缺失突变体。
六、转基因水稻的表型观察
1、OsCIP3超表达株系表型观察
将编号为OE8/ZH11和OE14/ZH11的T2代转OsCIP3水稻种子和ZH11(WT)种子以及空载对照植株(导入pUN1301载体)种子,30℃水中浸泡3天,萌发后,置于96孔板上,木村B培养液(Kato-Noguchi et al.,2005)里,在人工气候室中(光强为10000μmol/m2/s,光照时间为14h/d,温度为30℃)培养至3叶期;再将3叶期幼苗放在4℃的低温水浴锅中处理84小时,然后转移回人工气候室恢复生长一个月,照相、统计存活率。每个株系32株,实验重复三次,结果取平均值。低温水浴鉴定方法参考(刘栋峰,唐永严,雒胜韬,罗伟,李志涛,种康,徐云远,植物学报,2019,54(4):509-514)。
照相结果如图3中A和B所示,低温处理前,野生型水稻(ZH11)与超表达株系并没有明显的表型不同;当4℃处理84时,恢复生长一个月后,OsCIP3超表达水稻对低温胁迫的耐受性与野生型水稻(ZH11)具有显著的差异。
对该结果的存活率统计如图3中C所示,可以看出,4℃处理完又恢复生长一个月以后,OE8/ZH11和OE14/ZH11的T2代转OsCIP3水稻的存活率分别为51.7%和44.7%,而ZH11存活率为9.06%。空载对照的植株表型和存活率与野生型基本一致,无统计学差异。
可以看出,低温处理后超表达OsCIP3水稻的存活率较野生型明显升高,说明过超表达OsCIP3水稻对低温耐受性增强。
2、OsCIP3突变体株系表型观察
将oscip3-2/ZH11与oscip3-3/ZH11的T2代种子和对应的野生型种子以及空载对照植株(导入BGK03载体)种子,30℃水中浸泡3天,萌发后,置于96孔板上,木村B培养液里,在人工气候室中(光强为10000μmol/m2/s,光照时间为14h/d,温度为30℃)培养至3叶期;再将oscip3-2、oscip3-3以及ZH11的3叶期幼苗放在4℃的低温水浴锅中处理72h,然后转移回人工气候室恢复生长一个月,照相、统计存活率。每个株系32株,实验重复三次,结果取平均值。
照相结果如图4中A和B所示,低温处理前,野生型水稻与突变体株系并没有明显的表型差异;当对oscip3-2、oscip3-3以及ZH11的3叶期幼苗放在4℃的低温水浴锅中处理72h并恢复生长一个月以后,oscip3-2与oscip3-3突变体对低温胁迫的耐受性与野生型水稻均具有显著的差异。
对该结果的存活率统计如图4中C所示,4℃处理完又恢复生长一个月以后,oscip3-2与oscip3-3存活率分别0.4%以及0.62%,而对应的ZH11的存活率分别为34%及40.37%。空载对照的植株表型和存活率与野生型基本一致,无统计学差异。
上述结果表明,低温处理后oscip3突变体水稻的存活率较野生型明显降低,说明oscip3突变体对低温耐受性明显降低。
上述木村B培养液组成如下(Kato-Noguchi et al.,2005):
A液母液:1L(200х)
B液母液:1L(200х)
Ca(NO3)2.4H2O 17.235g
EDTA-Fe母液:1L(1000х)
溶解5.57g FeSO4.7H2O于200mL蒸馏水中,溶解7.45g Na2EDTA于200mL蒸馏水中,加热Na2EDTA溶液,加入FeSO4.7H2O溶液,不断搅拌,冷却后定容至1L。
微量元素母液:1L(1000х)
硅酸钠:每L木村B培养液用量100~300mg
1mol/L HCl:8.17mL 37%HCl用蒸馏水稀释至1000mL。
用1mol/L HCl调木村B培养液pH值为5.8。
实际应用中,取5ml A液母液、5ml B液母液、1ml EDTA-Fe母液、1ml微量元素母液、100~300mg硅酸钠混合加蒸馏水稀释至1L,用1mol/L HCl调木村B培养液pH值为5.8,得到1L木村B培养液。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
<110> 中国科学院植物研究所
<120> 通过过表达水稻中的锌指蛋白OsCIP3增强其低温耐受性
<130> GNCLN201476
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 522
<212> PRT
<213> Oryza sativa L.
<400> 1
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<210> 2
<211> 1569
<212> DNA
<213> Oryza sativa L.
<400> 2
atggacagtg gcttgggaag aagttcagag acatccttga aagccttgcc atcaatggca 60
agtaatgcaa caaggaatac tgatcctgac caacagggtg ttcgattcag ttccatggac 120
cagcctccat gttttgcaag acctggtcaa tcgttccctg cttttcctcc actctttggg 180
gttcagtctt ccagcttgta tttacctgat gacattgaag ctaaaatcgg taaccagttc 240
gaatcaaatc cttccccgaa taatcctaca atggattggg accctcaggc aatgctgagc 300
aacttatcct tccttgagca gaagatcaag caggtaaaag acatcgtgca gtctatgagt 360
aatcgtgaga gccaagttgc tggtggttcc agcgaggcac aagcaaagca gcagcttgtc 420
actgctgatc tcacttgtat tataattcag cttatttcaa cagctggttc cttgcttcct 480
tcgatgaaga acccaatcag cagcaacccg gcactcagac atctcagtaa cacactttgt 540
gctcctatga tcctgggcac caattgtaac ctgcgaccaa gcgcaaacga cgaagccaca 600
attcctgaca ttagcaagac ccatgactat gaggagctga tgaatagcct taatactact 660
caggcagaaa gcgatgagat gatgaactgc caaaatcctt gtggtgggga agggtcagaa 720
ccgattccaa tggaagacca tgatgtgaag gagagtgatg atggtggtga gagagagaat 780
ctcccccctg ggtcttatgt agtattgcaa ttagagaagg aggagatttt agcaccacat 840
actcacttct gcttgatctg tggcaagggt tttaaaagag atgctaatct taggatgcac 900
atgaggggcc atggagacga gtacaaaact gctgcagctc ttgcgaaacc ttcgaaagat 960
tctagcttag agtctgcacc agttacaagg tactcgtgcc catatgttgg ctgcaagcgg 1020
aacaaagagc acaagaagtt ccagcctctc aagacaatcc tgtgtgtgaa gaaccactac 1080
aagagaagcc actgtgacaa gagctacacc tgcagccgtt gcaacaccaa gaagttctca 1140
gttatcgcgg acttgaagac tcatgaaaag cactgtggcc gcgacaagtg gctatgctcg 1200
tgtggaacta ccttctcaag aaaagacaag ttatttgggc atgttgctct tttccaaggg 1260
cacacgcctg cactccctat ggatgatatc aaagtaacag gagcatcaga gcaacctcaa 1320
ggcagcgagg cgatgaacac catggtgggg agcgctgggt ataacttccc cggtagctca 1380
tcggacgaca tcccaaatct tgacatgaag atggctgatg atccacgcta tttctcgcca 1440
ttgagctttg atccttgctt cggtgggctt gatgacttca ctcgacctgg atttgacatc 1500
tctgagaatc ccttctcctt cttgccctca ggatcatgca gcttcgggca gcagaacgga 1560
gacagctga 1569
<210> 3
<211> 2291
<212> DNA
<213> Artificial sequence
<400> 3
ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60
agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120
tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180
tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240
gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300
ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360
gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420
agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480
taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540
aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600
cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660
cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720
acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780
ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840
gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900
ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960
cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020
ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080
ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140
ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200
gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260
agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320
atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380
tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440
tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500
aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560
cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620
gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680
acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740
gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800
tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860
tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920
catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980
gttacttctg caggtcgact ctagaggatc cccgggtacc gagctcgaat ttccccgatc 2040
gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 2100
ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 2160
cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 2220
tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 2280
tactagatcg g 2291
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence
<400> 4
tcaggcaatg ctgagcaac 19
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence
<400> 5
ccctgggtct tatgtagtat tg 22
Claims (10)
1.OsCIP3蛋白或其相关生物材料在调控植物低温耐受性中的应用;
所述相关生物材料为能够表达所述OsCIP3蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系;
所述OsCIP3蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
2.根据权利要求1所述的应用,其特征在于:所述OsCIP3蛋白在所述植物中的表达量和/或活性提高,所述植物的低温耐受性提高;和/或
所述OsCIP3蛋白在所述植物中的表达量和/或活性降低,所述植物的低温耐受性降低。
3.一种培育低温耐受性改变的植物品种的方法,为如下方法A1或方法A2:
方法A1:一种培育低温耐受性提高的植物品种的方法,包括使受体植物中OsCIP3蛋白的表达量和/或活性提高的步骤;
方法A2:一种培育低温耐受性降低的植物品种的方法,包括使受体植物中OsCIP3蛋白的表达量和/或活性降低的步骤;
所述OsCIP3蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
4.一种培育低温耐受性改变的转基因植物的方法,为如下方法B1或方法B2:
方法B1:一种培育低温耐受性提高的转基因植物的方法,包括如下步骤:向受体植物中导入能够表达OsCIP3蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比低温耐受性提高;
方法B2:一种培育低温耐受性降低的转基因植物的方法,包括如下步骤:对受体植物中能够表达OsCIP3蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比低温耐受性降低;
所述OsCIP3蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质;
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
5.根据权利要求4所述的方法,其特征在于:在所述方法B1中,能够表达所述OsCIP3蛋白的核酸分子是通过重组载体的形式导入所述受体植物中的。
6.根据权利要求5所述的方法,其特征在于:所述重组载体中启动能够表达所述OsCIP3蛋白的核酸分子转录的启动子为玉米泛素启动子。
7.根据权利要求1-6中任一所述的应用或方法,其特征在于:能够表达所述OsCIP3蛋白的核酸分子为如下任一所述的DNA分子:
(a1)SEQ ID No.2所示的DNA分子;
(a2)在严格条件下与(a1)限定的DNA分子杂交且编码所述OsCIP3蛋白的DNA分子;
(a3)与(a1)-(a2)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述OsCIP3蛋白的DNA分子。
8.根据权利要求1-7中任一所述的应用或方法,其特征在于:所述植物为单子叶植物。
9.根据权利要求8所述的应用或方法,其特征在于:所述单子叶植物为禾本科植物。
10.根据权利要求9所述的应用或方法,其特征在于:所述禾本科植物为水稻。
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